For the understanding of specific properties and functions in various types of biological macromolecules such as protein and complexes thereof, detailed steric structures thereof are indispensable information. From the basic biochemical standpoint, for example, information on the three-dimensional structure of protein or the like becomes the basis to understand the mechanism of functional expression in the biochemistry system with an enzyme or hormone. Particularly in the fields of pharmacy, genetic engineering, and chemical engineering among the industrial circles, the three-dimensional structure provides information indispensable for rational molecular design to facilitate drug designing, protein engineering, biochemical synthesis and the like.
As to the method of obtaining a three-dimensional steric structure of biological macromolecules at the atomic level, X-ray crystal structural analysis is the most cogent and precise means at present. The speed for analysis is significantly improving by virtue of the drastic increasing in the processing speed of computers in addition to reduction in the time for measurement and improvement in the measuring accuracy resulting from the recent hardware improvement of X-ray light sources and analyzers. It is expected that the three-dimensional structure will be clarified mainly depending upon this method.
In order to determine the three-dimensional structure of biological macromolecules by X-ray crystal structural analysis, it is essential to crystallize the target substance after extraction and purification. At present, there is neither technique nor apparatus that can be applied to every substance to achieve crystallization. In a conventional crystallization process, trial and error has been repeated relying on intuition and experience. In order to obtain a crystal of a biological macromolecule, the process for crystal growth has been required great numbers of experimental conditions, and it has been a serious bottleneck in the field of X-ray crystallographic analysis.
In a conventional process for crystallization of a biological macromolecule such as protein, a treatment for eliminating a solvent from an aqueous or non-aqueous solution containing the macromolecule is basically carried out, so that the resulting supersaturated state can reduce the solubility, leading to crystal growth. That is similar to the crystal growth process for low molecular weight compounds such as inorganic salts. As typical methods thereof, a batch method, dialysis, and diffusion are known. These methods are used depending upon the type, quantity, property, and the like of the sample.
In the batch method, a precipitant for eliminating the water of hydration is directly added to a solution containing a biological macromolecule to reduce its solubility, leading to its conversion into a solid phase. In this method, solid ammonium sulfate, for example, is often used. This method is disadvantageous in that a large amount of sample solution is required, fine adjustment of the salt concentration and pH is difficult, skill is required in operation, and reproducibility is low. In the dialysis method that is a improved one to eliminate some faults of the batch method, a solution including a biological macromolecule is filled in a sealed dialytic tube, and the pH or the like of the dialytic tube surrounding liquid such as a buffer solution is altered to induce crystallization. This method allows adjustment of the salt concentration and difference in pH of the inner and outer solutions at an arbitrary speed to facilitate the research for the crystallization conditions. One of the diffusion methods such as a gas-liquid phase diffusion method is shown in FIG. 39. A droplet 397 of a sample solution is placed on sample holders 393a and 393b. The droplet 397 and precipitant solutions 394a and 394b are retained in containers 391a and 391b, respectively, sealed by a cap 392, whereby the volatile components of the droplet and the precipitant solutions are vaporized into equilibrium. More preferable conditions can be obtained using different precipitants in a plurality of containers as shown in the drawing. In a liquid-liquid phase diffusion method, a droplet 407 of the mother liquor including the target substance and a droplet 404 of a precipitant are placed approximately 5 mm apart on a substrate 401, as shown in FIGS. 40(a) and 40(b). A thin liquid channel 406 is formed between the droplets by the tip of a needle or the like. Mutual diffusion through the channel 406 promotes crystallization. These diffusion methods are advantageous over the batch method in that the required amount of solution is extremely small.
However, there are still various problems as described above in the crystallization process of biological macromolecules such as proteins.
Many biological macromolecules do not have good crystallinity, so that a single crystal of them cannot be easily formed in a large size. This is probably because of the fact that the biological macromolecules generally having a great molecular weight are highly susceptible to gravity, causing convection in the solution (cf, F. Rosenberger, J. Cryst. Growth, 76, 618 (1986)). The small crystal nucleus of the biological macromolecules precipitates by its own weight, whereby convection occurs around the molecules or the crystal nucleus in the solution. The reduction in the concentration of the molecules also causes local convection at the surface of the grown crystal in the solution. The generated convection moves the grown crystal in the solution. Particularly around the crystal, the molecular supply layer is significantly reduced by the convection in the solution. Accordingly, the crystal growth rate is reduced, and anisotropic growth occurs at the crystal plane, so that crystallization is inhibited.
The crystal of biological macromolecules may contain a larger amount (.gtoreq.50% by volume) of solvent (mainly water from mother liquor) as compared with the crystal of other substances. The solvent is disorderly and readily movable in the intermolecular clearances of the crystal. Though the size of the molecules is relatively large, there is little packing contact between the molecules in a wide range of the crystal, and the weak bond by the van der Waals force between the molecules or the hydrogen bond via the water molecule simply contributes to the contact. These are also related to the inhibited crystallization.
The biological macromolecules may be very sensitive to the conditions for crystallization. Although the biological macromoleculars may be stabilized in the solvent by the interaction between the molecular surfaces, the charge distribution on the molecular surface and particularly the conformation of amino acids around the molecular surface may significantly vary with the environmental factor such as pH, ion strength, and temperature of the solution, the type and dielectric constant of the buffer solution, and the like. Therefore, the crystallization is a multi-parameter process with a complicated combination of various conditions. Thus, a universal crystallization technique that can be effective for any substance is under development. Especially, crystallization of hydrophobic proteins that may have more biochemical interest as compared with water-soluble proteins is very difficult. Only a few cases have been successful in crystallization of hydrophobic proteins and the analysis thereof with high resolution.
The resulting amount of biological macromolecules may often be quite small. For example, when a protein such as an enzyme is extracted from cells or the like and purified, the original content thereof may often be small and therefore the finally obtained sample for crystallization may be extremely small. In general, crystallization of biological macromolecules may require their concentration to be about 50 mg/ml in the solution. Therefore, each of repeated experiments (screening) under various conditions for crystallization must be carried out with a minimized amount of solution.
As mentioned above, the diffusion method does not require a great amount of sample. However, in order to obtain a crystal of good quality, the optimum conditions for crystallization must be obtained by altering the concentration of salt in the precipitant, pH or the like over a wide range. In such a case, trial and error is always involved for the adjustment of the conditions. In addition, the material such as a glass substrate on which the droplet of the sample is formed may often induce an undesired large amount of crystal nuclei. To prevent this event, the material surface should be processed by polishing, water-repellent finish or the like.
Although crystallization of biological macromolecules such as proteins and complexes thereof is an important process in science and industry, the conventional process for crystallization has always involved trial and error. The process for crystallization is a greatest barrier against the X-ray crystal structural analysis. Accordingly, desired is a crystallization technique applicable to any molecule based on a universal principle of crystallization.
Japanese Patent Laying-Open No. 4-182398 discloses an improved diffusion method. In this method, a biological macromolecular solution and a crystallization agent are allowed to stand in contact with each other at their surfaces, and then the container holding the biological macromolecule solution is divided into some parts by insertion of partitions, after the crystallization agent is allowed to diffuse into the biological macromolecular solution to a certain extent. From the obtained parts, a part or parts, which have more preferable conditions for crystallization, are selected. Japanese Patent Laying-Open No. 6-321700 discloses a method of preventing convection in the solution for crystallization. According to this method, acrylic amide, agarose or the like is added to the buffer in which the substance to be crystallized is dissolved, so that the substance is fixed in the resulting gel. The target substance is set in a supersaturated state in the gel by cooling, heating, or diffusing the precipitant introduced into the gel, for crystallization.
Over the above-mentioned conventional art, the inventor has developed a unique technique for crystallization as disclosed in Japanese Patent Laying-Open No. 8-294601. According to the developed method, crystallization of biological macromolecules such as proteins can be controlled on the surface of a solid-state component having the electrical state controlled under the control of the valence electrons, for example, on the surface of a silicon substrate doped with a certain type of impurity in a given concentration.